Advances in molecular biology have allowed the development of biological agents useful in modulating protein activity or nucleic acid expression, respectively. Many of these advances are based on the knowledge of the primary sequence of the molecule to be modulated. For example, the knowledge of the nucleic acid-sequence of DNA or RNA allows the development of antisense or ribozyme molecules. Similarly, the knowledge of the primary sequence allows for the identification of sequences that may be useful in creating monoclonal antibodies. Often, however, the knowledge of the primary sequence of a protein is insufficient to allow development of therapeutic or diagnostic molecules due to the secondary, tertiary or quartenary structure of the protein from which the primary sequence is obtained. In addition, mere knowledge of the primary sequence of a protein is insufficient to allow development of novel enzymes that facilitate the production of novel products or production of known reaction products under desired conditions (i.e., conditions under which such conversion does not ordinarily occur). The process of designing potent and specific inhibitors, activators, or novel proteins has improved with the arrival of techniques for determining the three-dimensional structure of an enzyme or polypeptide, whose activity substrate specificity or resulting enzymatic product one desires to modulate.
Methylation of oxygen (O-methylation), nitrogen (N-methylation), and carbon (C-methylation) is a universal process critical to all organisms. In plants, the O-methylation patterns of polyhydroxylated small molecules are of particular utility and importance. These site-specific reactions are crucial to determining final product distribution via multiple branched biosynthetic pathways using the same or similar intermediates and substrates. For example, the secondary metabolic pathway of phenylpropanoid biosynthesis utilizes cinnamate and acetate units to construct a diverse set of hydroxylated and polycyclic aromatic compounds which are used for regulatory, structural, and functional purposes in plants including protection against UV photodamage, pigmentation, fertilization, signaling, gene induction, anti-microbial defense, chemoattraction, and structural support. Additionally, phytochemicals mediate important biological activities in mammals. For example, isoflavones such as formononetin, (7-hydroxy-4′-methoxyisoflavone), daidzein (4′,7-dihydroxyisoflavone), and genistein (4′,5,7-trihydroxyisoflavone) possess phytoestrogenic and anti-oxidant activity. Consumption of a diet high in flavonoid and isoflavonoid compounds is salutary in reducing the incidence of certain types of cancer and lowering the risk for cardiovascular disease. Site specific methylation of flavonoid and isoflavonoid derivatives modulates their in vivo activity by limiting the number of reactive hydroxyl groups, altering the solubility properties of the resulting products, and ultimately determining whether a particular small molecule will interact with cellular receptors.
O-methylation is a common downstream modification. Although several S-adenosyl-L-methionine (SAM)-dependent O-methyltransferase (OMT) genes have been found in polyketide synthase (PKS) gene clusters (Decker, H. et al. J. Bacteriol. (1993) 175:3876-3886), their specificities have not been systematically studied as yet. It is suspected that some of them could be useful for combinatorial biosynthesis. For instance, O-11-methylation occurs in several members of the anthracycline, tetracenomycin, and angucycline classes of aromatic polyketides.
An improvement in the understanding of the structure/function of these enzymes would allow for a number of advances in the art, e.g., the exploitation of the synthetic capabilities of known enzymes for production of useful new chemical compounds, for the creation of novel non-native enzymes having new synthetic capabilities and the like. The present invention addresses this and related needs.